EP2079834A1 - Adn polymérases mutantes et procédés associés - Google Patents

Adn polymérases mutantes et procédés associés

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Publication number
EP2079834A1
EP2079834A1 EP07819100A EP07819100A EP2079834A1 EP 2079834 A1 EP2079834 A1 EP 2079834A1 EP 07819100 A EP07819100 A EP 07819100A EP 07819100 A EP07819100 A EP 07819100A EP 2079834 A1 EP2079834 A1 EP 2079834A1
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EP
European Patent Office
Prior art keywords
dna polymerase
polymerase
dna
amino acid
primer
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Granted
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EP07819100A
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German (de)
English (en)
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EP2079834B1 (fr
Inventor
Keith A. Bauer
Edward S. Smith
David Harrow Gelfand
Ellen Fiss
Shawn Suko
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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Publication of EP2079834A1 publication Critical patent/EP2079834A1/fr
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241Nucleotidyltransferases (2.7.7)
    • C12N9/1252DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241Nucleotidyltransferases (2.7.7)
    • C12N9/1276RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/34Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present invention lies in the field of DNA polymerases and their use in various applications, including nucleic acid primer extension and amplification.
  • DNA polymerases are responsible for the replication and maintenance of the genome, a role that is central to accurately transmitting genetic information from generation to generation.
  • DNA polymerases function in cells as the enzymes responsible for the synthesis of DNA. They polymerize deoxyribonucleoside triphosphates in the presence of a metal activator, such as Mg 2+ , in an order dictated by the DNA template or polynucleotide template that is copied.
  • a metal activator such as Mg 2+
  • DNA polymerases participate in a spectrum of DNA synthetic processes including DNA replication, DNA repair, recombination, and gene amplification. During each DNA synthetic process, the DNA template is copied once or at most a few times to produce identical replicas.
  • DNA replication can be repeated many times such as, for example, during polymerase chain reaction (see, e.g., U.S. Patent No. 4,683,202 to Mullis).
  • thermostable DNA polymerases could be obtained from bacteria that grow at elevated temperatures, and that these enzymes need to be added only once (see U.S. Patent No. 4,889,818 to Gelfand and U.S. Patent No. 4,965,188 to Mullis). At the elevated temperatures used during PCR, these enzymes are not irreversibly inactivated. As a result, one can carry out repetitive cycles of polymerase chain reactions without adding fresh enzymes at the start of each synthetic addition process.
  • DNA polymerases particularly thermostable polymerases
  • thermostable polymerases are the key to a large number of techniques in recombinant DNA studies and in medical diagnosis of disease.
  • a target nucleic acid sequence may be only a small portion of the DNA or RNA in question, so it may be difficult to detect the presence of a target nucleic acid sequence without amplification.
  • the overall folding pattern of polymerases resembles the human right hand and contains three distinct subdomains of palm, fingers, and thumb. (See Beese et al, Science 260:352-355, 1993); Patel et al, Biochemistry 34:5351-5363, 1995). While the structure of the fingers and thumb subdomains vary greatly between polymerases that differ in size and in cellular functions, the catalytic palm subdomains are all superimposable.
  • motif A which interacts with the incoming dNTP and stabilizes the transition state during chemical catalysis, is superimposable with a mean deviation of about one A amongst mammalian pol ⁇ and prokaryotic pol I family DNA polymerases (Wang et al, Cell 89:1087-1099, 1997). Motif A begins structurally at an antiparallel ⁇ -strand containing predominantly hydrophobic residues and continues to an ⁇ -helix. The primary amino acid sequence of DNA polymerase active sites is exceptionally conserved. In the case of motif A, for example, the sequence DYSQIELR is retained in polymerases from organisms separated by many millions years of evolution, including, e.g. , Thermus aquaticus, Chlamydia trachomatis, and Escherichia co/7. Taken together, these observations indicate that polymerases function by similar catalytic mechanisms.
  • DNA polymerases In addition to being well-conserved, the active site of DNA polymerases has also been shown to be relatively mutable, capable of accommodating certain amino acid substitutions without reducing DNA polymerase activity significantly. (See, e.g., U.S. Patent No. 6,602,695 to Patel et al.) Such mutant DNA polymerases can offer various selective advantages in, e.g., diagnostic and research applications comprising nucleic acid synthesis reactions. Thus, there is a need in the art for identification of amino acid positions amenable to mutation to yield improved polymerase activity, including, for example, improved extension rates, reverse transcription efficiency, or amplification ability. The present invention, as set forth herein, meets these and other needs. BRIEF SUMMARY OF THE INVENTION
  • the present invention provides a mutant DNA polymerase having improved enzyme activity relative to the corresponding unmodified polymerase and which is useful in a variety of nucleic acid synthesis applications, hi some embodiments, the polymerase comprises an amino acid sequence having at least one of the following motifs in the polymerase domain:
  • the polymerase has an improved nucleic acid extension rate and/or an improved reverse transcription efficiency relative to an otherwise identical polymerase
  • X a8 is an amino acid selected from Q, T, M, G or L
  • X b8 is an amino acid selected from D, E or N
  • X c6 is an amino acid selected from S, A, V, or G (i.e., a reference polymerase).
  • X a8 is Q, T, M, G or L
  • X b8 is D, E or N
  • X 04 is I or L
  • X 06 is S, A, V, or G (SEQ ID NOS :23 and 24).
  • X b8 is D, E or N (SEQ ID NOS :25 and 26).
  • X a8 is a D- or L-amino acid selected from the group consisting of: A, C, D, E, F, H, I, K, N, P, R, S, V, W, Y (SEQ ID NO:27), and analogs thereof.
  • X 38 is an amino acid selected from the group consisting of: R, K and N (SEQ ID NO:28).
  • X a8 is Arginine (R) (SEQ ID NO:29).
  • X b8 is D- or L-amino acid selected from the group consisting of: A, C, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (SEQ ID NO:30), and analogs thereof.
  • X b8 is an amino acid selected from the group consisting of: G, A, S, T, R, K, Q, L, V and I (SEQ ID NO:31).
  • X b8 is an amino acid selected from the group consisting of: G, T, R, K and L (SEQ ID NO:32).
  • X 04 is a D- or L-amino acid selected from the group consisting of: A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T, V, W, Y (SEQ ID NO:33), and analogs thereof.
  • X 04 is an amino acid selected from the group consisting of F and Y (SEQ ID NO:34).
  • X c4 is phenylalanine (F) (SEQ ID NO:35).
  • X 06 is an amino acid selected from the group consisting of C, D, E, F, H, I, K, L, M, N, P, Q, R, T, W and Y (SEQ ID NO:36).
  • X 06 is an amino acid selected from the group consisting of F and Y (SEQ ID NO.37).
  • X 06 is phenylalanine (F) (SEQ ID NO:38).
  • the improved polymerases e.g., Z05 or CS5/CS6 that comprise at least one of Arginine (R) at position X a8 ; Glycine (G) at position X b8 ; Phenylalanine (F) at position X 04 ; and/or Phenylalanine (F) at position X 06 (SEQ ID NOS:39-68).
  • the DNA polymerases of the invention are modified versions of an unmodified polymerase.
  • the polymerase includes an amino acid sequence having the following motifs in the polymerase domain:
  • X 0I is G, N or D
  • X 02 is W or H
  • X c3 is W, A, L or V
  • X 04 is I or L
  • X 05 is V, F or L
  • X 06 is S, A, V or G
  • X 07 is A or L.
  • thermostable polymerases including wild-type or naturally occurring thermostable polymerases from various species of thermophilic bacteria, as well as thermostable polymerases derived from such wild-type or naturally occurring enzymes by amino acid substitution, insertion, or deletion, or other modification.
  • exemplary unmodified forms of polymerase include, e.g., CS5, CS6 or Z05 DNA polymerase, or a functional DNA polymerase having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • unmodified polymerases include, e.g., DNA polymerases from any of the following species of thermophilic bacteria (or a functional DNA polymerase having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to such a polymerase): Thermotoga maritima; Thermus aquaticus; Thermus thermophilus; Thermus flavus; Thermus filiformis; Thermus sp. spsl7; Thermus sp. Z05; Thermotoga neopolitana; Thermosipho africanus; Thermus acidophilus or Bacillus caldotenax.
  • Suitable polymerases also include those having reverse transcriptase (RT) activity and/or the ability to incorporate unconventional nucleotides, such as ribonucleotides or other 2'- modified nucleotides.
  • RT reverse transcriptase
  • the unmodified form of the polymerase comprises a chimeric polymerase.
  • the unmodified form of the chimeric polymerase is CS5 DNA polymerase (SEQ ID NO: 18), CS6 DNA polymerase (SEQ ID NO: 19), or a polymerase having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the CS5 DNA polymerase or the CS6 DNA polymerase.
  • the unmodified form of the chimeric polymerase includes one or more amino acid substitutions relative to SEQ ID NO: 18 or SEQ ID
  • the unmodified form of the mutant polymerase can be G46E CS5; G46E L329A CS5; G46E E678G CS5; or G46E L329A E678G CS5.
  • these unmodified forms are substituted to provide a mutant polymerase including one or more amino acid substitutions selected from S671F, D640G, Q601R, and I669F.
  • the mutant DNA polymerase can be any one of the following: G46E S671F CS5; G46E D640G CS5; G46E Q601R CS5; G46E I669F CS5; G46E D640G S671F CS5; G46E L329A S671F CS5; G46E L329A D640G CS5; G46E L329A Q601R CS5; G46E L329A I669F CS5; G46E L329A D640G S671F CS5; G46E S671F E678G CS5; G46E D640G E678G CS5; G46E Q601R E678G CS5; G46E I669F E678G CS5; G46E L329A S671F E678G CS5; G46E L329A D640G E678G CS5; G46E L329A D640G
  • the polymerase is a CS5 polymerase (SEQ ID NO: 15), a CS6 polymerase (SEQ ID NO: 16) or a Z05 polymerase (SEQ ID NO:6), wherein X b8 is an amino acid selected from the group consisting of: G, T, R, K and L.
  • the CS5 or CS6 polymerase can be selected from the following: D640G, D640T, D640R, D640K and D640L.
  • the Z05 polymerase can be selected from the group consisting of: D580G, D580T, D580R, D580K and D580L.
  • the mutant polymerase can include other, non-substitutional modifications.
  • One such modification is a thermally reversible covalent modification that inactivates the enzyme, but which is reversed to activate the enzyme upon incubation at an elevated temperature, such as a temperature typically used for primer extension.
  • the mutant polymerase comprising the thermally reversible covalent modification is produced by a reaction, carried out at alkaline pH at a temperature that is less than about 25°C, of a mixture of a thermostable DNA polymerase and a dicarboxylic acid anhydride having one of the following formulas I or II:
  • Ri and R 2 are hydrogen or organic radicals, which may be linked;
  • Ri and R 2 are organic radicals, which may linked, and the hydrogens are cis.
  • the unmodified form of the polymerase is G64E CS5.
  • the extension rate is determined using a single-stranded DNA as a template (e.g, M13mpl8, HIV), primed with an appropriate primer (e.g., a polynucleotide of the nucleic acid sequence
  • the extension rate of a polymerase of the invention can be compared to the extension rate of a reference polymerase (e.g., a naturally occurring or unmodified polymerase), over a preselected unit of time, as described herein.
  • a reference polymerase e.g., a naturally occurring or unmodified polymerase
  • the present invention provides a recombinant nucleic acid encoding a mutant DNA polymerase as described herein, a vector comprising the recombinant nucleic acid, and a host cell transformed with the vector.
  • the vector is an expression vector.
  • Host cells comprising such expression vectors are useful in methods of the invention for producing the mutant polymerase by culturing the host cells under conditions suitable for expression of the recombinant nucleic acid.
  • the polymerases of the invention may be contained in reaction mixtures and/or kits.
  • the embodiments of the recombinant nucleic acids, host cells, vectors, expression vectors, eraction mixtures and kits are as described above and herein.
  • a method for conducting primer extension generally includes contacting a mutant DNA polymerase of the invention with a primer, a polynucleotide template, and free nucleotides under conditions suitable for extension of the primer, thereby producing an extended primer.
  • the polynucleotide template can be, for example, an RNA or DNA template.
  • the free nucleotides can include unconventional nucleotides such as, e.g., ribonucleotides and/or labeled nucleotides.
  • the primer and/or template can include one or more nucleotide analogs.
  • the primer extension method is a method for polynucleotide amplification that includes contacting the mutant DNA polymerase with a primer pair, the polynucleotide template, and the free nucleotides under conditions suitable for amplification of the polynucleotide.
  • the present invention also provides a kit useful in such a primer extension method.
  • the kit includes at least one container providing a mutant DNA polymerase as described herein.
  • the kit further includes one or more additional containers providing one or more additional reagents.
  • the one or more additional containers provide free nucleotides; a buffer suitable for primer extension; and/or a primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template.
  • reaction mixtures comprising the polymerases of the invention.
  • the reactions mixtures can also contain a template nucleic acid (DNA and/or RNA), one or more primer or probe polynucleotides, free nucleotides (including, e.g., deoxyribonucleotides, ribonucleotides, labeled nucleotides, unconventional nucleotides), buffers, salts, labels (e.g., fluorophores).
  • amino acid refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein.
  • amino acid includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (GIn or Q), glutamic acid (GIu or E), glycine (GIy or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine
  • amino acid also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and/or backbones), and amino acid analogs.
  • an amino acid is typically an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups.
  • exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups.
  • amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal- containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, carbon-linked sugar- containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.
  • mutant in the context of DNA polymerases of the present invention, means a polypeptide, typically recombinant, that comprises one or more amino acid substitutions relative to a corresponding, functional DNA polymerase.
  • mutant form in the context of a mutant polymerase, is a term used herein for purposes of defining a mutant DNA polymerase of the present invention: the term “unmodified form” refers to a functional DNA polymerase that has the amino acid sequence of the mutant polymerase except at one or more amino acid position(s) specified as characterizing the mutant polymerase.
  • reference to a mutant DNA polymerase in terms of (a) its unmodified form and (b) one or more specified amino acid substitutions means that, with the exception of the specified amino acid substitution(s), the mutant polymerase otherwise has an amino acid sequence identical to the unmodified form in the specified motif.
  • the polymerase may contain additional mutations to provide desired functionality, e.g., improved incorporation of dideoxyribonucleotides, ribonucleotides, ribonucleotide analogs, dye-labeled nucleotides, modulating 5 '-nuclease activity, modulating 3 '-nuclease (or proofreading) activity, or the like.
  • the unmodified form of a DNA polymerase is predetermined.
  • the unmodified form of a DNA polymerase can be, for example, a wild-type and/or a naturally occurring DNA polymerase, or a DNA polymerase that has already been intentionally modified.
  • An unmodified form of the polymerase is preferably a thermostable DNA polymerases, such as DNA polymerases from various thermophilic bacteria, as well as functional variants thereof having substantial sequence identity to a wild-type or naturally occurring thermostable polymerase
  • Such variants can include, for example, chimeric DNA polymerases such as, for example, the chimeric DNA polymerases described in U.S. Patent No. 6,228,628 and U.S. Application Publication No. 2004/0005599.
  • the unmodified form of a polymerase has reverse transcriptase (RT) activity.
  • thermostable polymerase refers to an enzyme that is stable to heat, is heat resistant, and retains sufficient activity to effect subsequent primer extension reactions and does not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids.
  • the heating conditions necessary for nucleic acid denaturation are well known in the art and are exemplified in, e.g., U.S. Patent Nos. 4,683,202, 4,683,195, and 4,965,188.
  • a thermostable polymerase is suitable for use in a temperature cycling reaction such as the polymerase chain reaction ("PCR").
  • Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity.
  • enzymatic activity refers to the catalysis of the combination of the nucleotides in the proper manner to form primer extension products that are complementary to a template nucleic acid strand.
  • Thermostable DNA polymerases from thermophilic bacteria include, e.g., DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilics, Thermusflavus, Thermus f ⁇ iformis, Thermus species spsl7, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, and Thermosipho africanus.
  • a "chimeric" protein refers to a protein whose amino acid sequence represents a fusion product of subsequences of the amino acid sequences from at least two distinct proteins.
  • a chimeric protein typically is not produced by direct manipulation of amino acid sequences, but, rather, is expressed from a "chimeric" gene that encodes the chimeric amino acid sequence.
  • an unmodified form of a mutant DNA polymerase of the present invention is a chimeric protein that consists of an amino-terminal (N-terminal) region derived from a Thermus species DNA polymerase and a carboxy-terminal (C-terminal) region derived from Tma DNA polymerase.
  • the N-terminal region refers to a region extending from the N- terminus (amino acid position 1) to an internal amino acid.
  • the C-terminal region refers to a region extending from an internal amino acid to the C-terminus.
  • “correspondence” to another sequence is based on the convention of numbering according to nucleotide or amino acid position number and then aligning the sequences in a manner that maximizes the percentage of sequence identity. Because not all positions within a given "corresponding region” need be identical, non-matching positions within a corresponding region may be regarded as "corresponding positions.” Accordingly, as used herein, referral to an "amino acid position corresponding to amino acid position [X]" of a specified DNA polymerase represents referral to a collection of equivalent positions in other recognized DNA polymerases and structural homologues and families.
  • amino acid positions are determined with respect to a region of the polymerase comprising one or more motifs of SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3, as discussed further herein.
  • Recombinant refers to an amino acid sequence or a nucleotide sequence that has been intentionally modified by recombinant methods.
  • recombinant nucleic acid herein is meant a nucleic acid, originally formed in vitro, in general, by the manipulation of a nucleic acid by endonucleases, in a form not normally found in nature.
  • an isolated, mutant DNA polymerase nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is typically distinguished from naturally occurring protein by at least one or more characteristics.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • host cell refers to both single-cellular prokaryote and eukaryote organisms (e.g. , bacteria, yeast, and actinomycetes) and single cells from higher order plants or animals when being grown in cell culture.
  • prokaryote and eukaryote organisms e.g. , bacteria, yeast, and actinomycetes
  • vector refers to a piece of DNA, typically double-stranded, which may have inserted into it a piece of foreign DNA.
  • the vector or may be, for example, of plasmid origin.
  • Vectors contain "replicon" polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell.
  • Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene.
  • the vector is used to transport the foreign or heterologous DNA into a suitable host cell.
  • the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated.
  • the vector can also contain the necessary elements that permit transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple copies of RNA.
  • Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Many molecules of mRNA and polypeptide encoded by the inserted DNA can thus be rapidly synthesized.
  • nucleotide in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, shall herein be understood to refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.
  • nucleic acid refers to a polymer that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or an analog thereof.
  • RNA ribose nucleic acid
  • DNA deoxyribose nucleic acid
  • polymers of nucleotides such as RNA and DNA, as well as synthetic forms, modified (e.g., chemically or biochemically modified) forms thereof, and mixed polymers (e.g., including both RNA and DNA subunits).
  • Exemplary modifications include methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g.,
  • nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can comprise other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497- 1500, 1991).
  • a nucleic acid can be or can include, e.g., a chromosome or chromosomal segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, the product of a polymerase chain reaction (PCR), an oligonucleotide, a probe, and a primer.
  • PCR polymerase chain reaction
  • a nucleic acid can be, e.g., single-stranded, double-stranded, or triple-stranded and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.
  • oligonucleotide refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides).
  • An oligonucleotide typically includes from about six to about 175 nucleic acid monomer units, more typically from about eight to about 100 nucleic acid monomer units, and still more typically from about 10 to about 50 nucleic acid monomer units (e.g., about 15, about 20, about 25, about 30, about 35, or more nucleic acid monomer units).
  • the exact size of an oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide.
  • Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (Meth. Enzymol 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J Am. Chem. Soc.
  • primer refers to a polynucleotide capable of acting as a point of initiation of template-directed nucleic acid synthesis when placed under conditions in which primer extension is initiated (e.g., under conditions comprising the presence of requisite nucleoside triphosphates (as dictated by the template that is copied) and a polymerase in an appropriate buffer and at a suitable temperature or cycle(s) of temperatures (e.g., as in a polymerase chain reaction)).
  • primers can also be used in a variety of other oligonuceotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes (e.g., nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), etc.).
  • a primer is typically a single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide).
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 40 nucleotides, more typically from 15 to 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • primer pair means a set of primers including a 5' sense primer (sometimes called “forward") that hybridizes with the complement of the 5' end of the nucleic acid sequence to be amplified and a 3' antisense primer (sometimes called “reverse") that hybridizes with the 3' end of the sequence to be amplified (e.g., if the target sequence is expressed as RNA or is an RNA).
  • a primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • nucleic acid bases nucleoside triphosphates, or nucleotides refers to those which occur naturally in the polynucleotide being described (i.e. , for DNA these are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and 7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATP can be utilized in place of dATP in in vitro DNA synthesis reactions, such as sequencing. Collectively, these may be referred to as dNTPs.
  • nucleic acid base nucleoside, or nucleotide
  • Certain unconventional nucleotides are modified at the T position of the ribose sugar in comparison to conventional dNTPs.
  • ribonucleotides are unconventional nucleotides as substrates for DNA polymerases.
  • unconventional nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing.
  • Exemplary terminator compounds include but are not limited to those compounds that have a 2',3' dideoxy structure and are referred to as dideoxynucleoside triphosphates.
  • the dideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP are referred to collectively as ddNTPs.
  • Additional examples of terminator compounds include 2'-PO 4 analogs of ribonucleotides ⁇ see, e.g., U.S. Application Publication Nos. 2005/0037991 and 2005/0037398).
  • Other unconventional nucleotides include phosphorothioate dNTPs ([[ ⁇ ]-S]dNTPs), 5'-[ ⁇ ]-borano-dNTPs, [ ⁇ ]- methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).
  • Unconventional bases may be labeled with radioactive isotopes such as 32 P, 33 P, or 35 S; fluorescent labels; chemiluminescent labels; bioluminescent labels; hapten labels such as biotin; or enzyme labels such as streptavidin or avidin.
  • Fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family, or dyes that are neutral in charge, such as dyes of the rhodamine family, or dyes that are positively charged, such as dyes of the cyanine family. Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE.
  • Dyes of the rhodamine family include Texas Red, ROX, Rl 10, R6G, and TAMRA.
  • Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, Rl 10, R6G, Texas Red and TAMRA are marketed by Perkin- Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen/Molecular Probes (Eugene, OR).
  • Dyes of the cyanine family include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).
  • percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • Sequences are "substantially identical" to each other if they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% identical. These definitions also refer to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • similarity in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • a conservative amino acid substitutions e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region
  • Sequences are "substantially similar” to each other if they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other.
  • this similarly exists over a region that is at least about 50 amino acids in length, or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J MoI. Evol. 35:351-360, 1987). The method used is similar to the method described by Higgins and Sharp (CABIOS 5:151-153, 1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
  • This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package (e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-95, 1984) or later).
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. MoI. Biol. 215:403-10, 1990), respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved vaiue; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.
  • nucleic acid extension rate refers the rate at which a biocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like) extends a nucleic acid (e.g., a primer or other oligonucleotide) in a template-dependent or template-independent manner by attaching (e.g., covalently) one or more nucleotides to the nucleic acid.
  • a biocatalyst e.g., an enzyme, such as a polymerase, ligase, or the like
  • a nucleic acid e.g., a primer or other oligonucleotide
  • certain mutant DNA polymerases described herein have improved nucleic acid extension rates relative to unmodified forms of these DNA polymerases, such that they can extend primers at higher rates than these unmodified forms under a given set of reaction conditions.
  • reverse transcription efficiency refers to the fraction of RNA molecules that are reverse transcribed as cDNA in a given reverse transcription reaction.
  • the mutant DNA polymerases of the invention have improved reverse transcription efficiencies relative to unmodified forms of these DNA polymerases. That is, these mutant DNA polymerases reverse transcribe a higher fraction of RNA templates than their unmodified forms under a particular set of reaction conditions.
  • Figure 1 depicts an amino acid sequence alignment of a region from the polymerase domain of exemplary thermostable DNA polymerases from various species of thermophilic bacteria: Thermus thermophilus (Tth) (SEQ ID NO:4), Thermus acidophilus (Tea) (SEQ ID NO:5), Thermus species Z05 (Z05) (SEQ ID NO:6), Thermus aquaticus (Taq) (SEQ ID NO.7), Thermus flavus (TfI) (SEQ ID NO:8), Thermus filiformis (Tfi) (SEQ ID NO:9), Thermus species spsl7 (Spsl7) (SEQ ID NO: 10), Thermotoga maritima (Tma) (SEQ ID NO:11), Thermotoga neapolitana (Tne) (SEQ ID NO: 12), Thermosipho africanus (Taf) (SEQ ID NO: 13), and Bacillus caldotenax (Bca
  • the amino acid sequence alignment also includes a region from the polymerase domain of representative chimeric thermostable DNA polymerases, namely, CS5 (SEQ ID NO: 15) and CS6 (SEQ ID NO: 16).
  • CS5 SEQ ID NO: 15
  • CS6 SEQ ID NO: 16
  • a sequence (Cons) SEQ ID NO: 17
  • the polypeptide regions shown comprise the amino acid motifs XXXXRXXXKLXXTYXX (SEQ ID NO: 1 ),
  • TGRLSSXXPNLQN SEQ ID NO:2
  • XXXXXXXD YS QIELR SEQ ID NO:3
  • Amino acid positions amenable to mutation in accordance with the present invention are indicated with an asterisk (*).
  • Gaps in the alignments are indicated with a dot (.).
  • Figure 2 A presents the amino acid sequence of the chimeric thermostable DNA polymerase CS5 (SEQ ID NO: 18).
  • Figure 2B presents a nucleic acid sequence encoding the chimeric thermostable DNA polymerase CS5 (SEQ ID NO:20).
  • Figure 3 A presents the amino acid sequence of the chimeric thermostable DNA polymerase CS6 (SEQ ID NO: 19).
  • Figure 3B presents a nucleic acid sequence encoding the chimeric thermostable DNA polymerase CS6 (SEQ ID NO21).
  • Figure 4 is a bar graph that shows the normalized extension rates of various mutants of a G46E L329A E678G (GLE) CS5 DNA polymerase.
  • the extension rate values obtained for the mutant polymerases are normalized relative to the value obtained for the GLE CS5 DNA polymerase, which is set to 1.00.
  • Figure 5 is a bar graph that shows the normalized extension rates of various mutants of a G46E L329A E678G (GLE) CS5 DNA polymerase.
  • the extension rate values obtained for the mutant polymerases are normalized relative to the value obtained for the GLE CS5 DNA polymerase, which is set to 1.00.
  • Figure 6 is a bar graph that shows the normalized extension rates of a Z05 DNA polymerase, a ⁇ Z05 DNA (dZ05 in Figure 6) polymerase (see, e.g., U.S. Pat. No. 5,455,170, entitled "MUTATED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM THERMUS SPECIES Z05" issued October 3, 1995 to Abramson et al. and U.S. Pat. No.
  • Figure 7 is a bar graph that shows the normalized extension rates of a Z05 DNA polymerase, a ⁇ Z05 (dZ05 in Figure 7) DNA polymerase, and various mutants of a G46E L329A (GL) CS5 DNA polymerase.
  • the extension rate values obtained for the mutant polymerases are normalized relative to the value obtained for a GLE CS5 DNA polymerase, which is set to 1.00.
  • Figure 8 is a plot that shows the extension rates of different DNA polymerases under varied salt (KOAc) concentrations.
  • the y-axis represents the extension rates (Arbitrary Units), while the x-axis represents KOAc concentration (raM).
  • the legend that accompanies the plot shows the DNA polymerase corresponding to each trace in the plot.
  • delta Z05 refers to ⁇ Z05 DNA polymerase and Z05 refers to Z05 DNA polymerase
  • Figure 9 is a plot that shows the extension rates of different DNA polymerases under varied salt (KOAc) concentrations.
  • the y-axis represents the extension rates (Arbitrary Units), while the x-axis represents KOAc concentration (mM).
  • the legend that accompanies the plot shows the DNA polymerase corresponding to each trace in the plot.
  • Figure 10 is a bar graph that shows the threshold cycle (Ct) values obtained for various mutant CS5 DNA polymerases in RT-PCRs.
  • Figure 11 is a bar graph that shows the threshold cycle (Ct) values obtained for various mutant CS5 DNA polymerases in Mg +2 -activated RT-PCRs having varied RT incubation times.
  • Figure 12 is a bar graph that shows the Ct values obtained for various mutant CS5 DNA polymerases in Mn +2 -activated RT-PCRs having varied RT incubation times.
  • FIGS 13 A and B are photographs of agarose gels that illustrate the ability of certain enzymes described herein to make full length amplicon under the various conditions involving ribonucleotides.
  • Figure 14A is a plot of delta Cts (y-axis) for the enzymes described with respect to Figures 13 A and B against various rATP conditions tested (y-axis), while Figure 14B is a plot of % rNTP incorporation (y-axis) for the enzymes described with respect to Figures 13 A and B against various rNTP conditions tested (y-axis).
  • FIGS 15A and B are photographs of agarose gels that illustrate the ability of certain enzymes described herein to make full length amplicon under the various conditions involving biotinylated ribonucleotides.
  • Figure 16A is a plot of delta Cts (y-axis) for the enzymes (x-axis) described with respect to Figures 15 A and B for various rCTP conditions tested (legend), while Figure 14B is a plot of delta Cts (y-axis) for those enzymes (x-axis) for various biotin labeled rCTP conditions tested (legend).
  • Figure 17 is a bar graph that shows the effect of enzyme concentration on threshold cycle (Ct) values in pyrophosphorolysis activated polymerization (PAP) reactions utilizing a G46E L329A E678G (GLE) CS5 DNA polymerase.
  • the y-axis represents Ct value, while the x-axis represents the enzyme concentration (nM).
  • the legend that accompanies the plot shows the number of copies of the template nucleic acid corresponding to each trace in the graph (no copies of the template nucleic acid (no temp), Ie 4 copies of the template nucleic acid (lE4/rxn), Ie 5 copies of the template nucleic acid (lE5/rxn), and Ie 6 copies of the template nucleic acid (lE6/rxn)).
  • Figure 18 is a bar graph that shows the effect of enzyme concentration on threshold cycle (Ct) values in pyrophosphorolysis activated polymerization (PAP) reactions utilizing a G46E L329A D640G S671F E678G (GLDSE) CS5 DNA polymerase.
  • the y-axis represents Ct value, while the x-axis represents the enzyme concentration (nM).
  • the legend that accompanies the plot shows the number of copies of the template nucleic acid corresponding to each trace in the graph (no copies of the template nucleic acid (no temp), Ie 4 copies of the template nucleic acid (lE4/rxn), Ie 5 copies of the template nucleic acid (lE5/rxn), and Ie 6 copies of the template nucleic acid (lE6/rxn)).
  • Figure 19 is a bar graph that shows the normalized extension rates of various mutants of a Thermus sp. Z05 DNA polymerase.
  • the x-axis represents also represents ESl 12 (E683R Z05 DNA polymerase; see, U.S. Pat. Appl. No.
  • Figure 20 is a photograph of a gel that shows the detection of PCR products from an analysis that involved PAP -related HIV DNA template titrations.
  • Figure 21 is a graph that shows threshold cycle (C T ) values observed for various mutant K-Ras plasmid template copy numbers utilized in amplifications that involved blocked or unblocked primers.
  • Figure 22 is a graph that shows threshold cycle (C T ) values observed for various enzymes and enzyme concentrations utilized in amplifications that involved a K-Ras plasmid template.
  • Figure 23 is a bar graph that shows data for PAP reverse transcription reactions on HCV RNA in which products of the cDNA reaction were measured using a quantitative PCR assay specific for the HCV cDNA.
  • the y-axis represents Ct value, while the x-axis represents the Units of enzyme utilized in the reactions. As indicated, the enzymes used in these reactions were Z05 DNA polymerase (Z05) or blends of G46E L329A Q601R D640G S671F E678G (GLQDSE) and G46E L329A Q601R D640G S671F (GLQDS) CS5 DNA polymerases.
  • Figure 24 shows PCR growth curves of BRAF oncogene amplifications that were generated when bidirectional PAP was performed. The x-axis shows normalized, accumulated fluorescence and the y-axis shows cycles of PAP PCR amplification.
  • the present invention provides novel mutant DNA polymerases in which one or more amino acids in the polymerase domain have been mutated relative to a functional DNA polymerase.
  • the mutant DNA polymerases of the invention are active enzymes having improved rates of nucleotide incorporation relative to the unmodified form of the polymerase and, in certain embodiments, concomitant increases in reverse transcriptase activity and/or amplification ability.
  • the mutant DNA polymerases may be used at lower concentrations for superior or equivalent performance as the parent enzymes.
  • the mutant DNA polymerases described herein have improved thermostability relative to parent enzymes.
  • the mutant DNA polymerases are therefore useful in a variety of applications involving primer extension as well as reverse transcription or amplification of polynucleotide templates, including, for example, applications in recombinant DNA studies and medical diagnosis of disease.
  • Unmodified forms of DNA polymerases amenable to mutation in accordance with the present invention are those having a functional polymerase domain comprising the following amino acid motifs:
  • X 32 is GIn (Q) or Leu (L);
  • X 33 is GIn (Q), His (H) or GIu (E);
  • X 34 is Tyr (Y), His (H), or Phe (F);
  • X 36 is GIu (E), GIn (Q) or Lys (K);
  • X a7 is He (I), Leu (L) or Tyr (Y);
  • X a8 is GIn (Q), Thr (T), Met (M), GIy (G) or Leu (L);
  • X al is Lys (K) or GIn (Q);
  • X al2 is Ser (S) or Asn (N);
  • X al5 is He (I) or VaI (V); and
  • X a i 6 is GIu (E) or Asp (D);
  • X b7 is Ser (S) or Thr (T);
  • X b8 is Asp (D), GIu (E) or Asn (N);
  • Leu-Arg also referred to herein in the one-letter code as X c1 -X c2 -X c3 -X c4 -X c5 -X C e-X c7 -D-Y-S-Q-I-E-L-R (SEQ ID NO: 1
  • X c i is GIy (G), Asn (N), or Asp (D);
  • X c2 is Trp (W) or His (H);
  • X c3 is Trp (W), Ala (A), Leu (L) or VaI (V);
  • X c4 is He (I) or Leu (L);
  • X c5 is VaI (V), Phe (F) or Leu (L);
  • X c6 is Ser (S), Ala (A), VaI (V) or GIy (G);
  • X c7 is Ala (A) or Leu (L).
  • thermostable motifs are present within a region of about 100 amino acids in the active site of many Family A type DNA-dependent DNA polymerases, particularly thermostable
  • Figure 1 shows an amino acid sequence alignment of a region from the polymerase domain of DNA polymerases from several species of thermophilic bacteria: Thermotoga maritima, Thermus aquaticus, Thermus thermophilics, Thermus flavus, Thermus ⁇ liformis, Thermus sp. spsl7, Thermus sp. Z05, Thermotoga neopolitana, Thermosipho africanus, Bacillus caldotenax and Thermus caldophilus.
  • the amino acid sequence alignment shown in Figure 1 also includes a region from the polymerase domain of representative chimeric thermostable DNA polymerases. As shown, each of the motifs of SEQ ID NOs. 1, 2, and 3 is present in each of these polymerases, indicating a conserved function for these regions of the active site.
  • the unmodified form of the DNA polymerase is a wild-type or a naturally occurring DNA polymerase, such as, for example, a polymerase from any of the species of thermophilic bacteria listed above.
  • the unmodified polymerase is from a species of the genus Thermus.
  • the unmodified polymerase is from a thermophilic species other than Thermus. The full nucleic acid and amino acid sequence for numerous thermostable DNA polymerases are available.
  • Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus species Z05, Thermus species spsl7, Thermotoga ma ⁇ tima (Tma), and Thermosipho africanus (Taf) polymerase have been published in PCT International Patent Publication No. WO 92/06200.
  • the sequence for the DNA polymerase from Thermus flavus has been published in Akhmetzjanov and Vakhitov (Nucleic Acids Research 20:5839, 1992).
  • the sequence of the thermostable DNA polymerase from Thermus caldophilus is found in EMBL/GenBank Accession No. U62584.
  • thermostable DNA polymerase from Thermus filiformis can be recovered from ATCC Deposit No. 42380 using, e.g., the methods provided in U.S. Pat. No. 4,889,818, as weii as the sequence information provided in Table 1.
  • the sequence of the Thermotoga neapolitana DNA polymerase is from GeneSeq Patent Data Base Accession No. R98144 and PCT WO 97/09451.
  • the sequence of the thermostable DNA polymerase from Bacillus caldotenax is described in, e.g., Uemori et al. (J Biochem (Tokyo) 1 13(3):401-410, 1993; see also, Swiss-Prot database Accession No.
  • thermostable DNA polymerases for improvement of nucleic acid synthesis and amplification in vitro
  • 6,881,559 entitled "Mutant B-type DNA polymerases exhibiting improved performance in PCR” issued April 19, 2005 to Sobek et al.
  • 6,794,177 entitled "Modified DNA-polymerase from carboxydothermus hydrogenoformans and its use for coupled reverse transcription and polymerase chain reaction” issued September 21, 2004 to Markau et al.
  • 6,468,775 entitled "Thermostable DNA polymerase from carboxydothermus hydrogenoformans” issued October 22, 2002 to Ankenbauer et al.; and U.S.
  • suitable unmodified DNA polymerases also include functional variants of wild-type or naturally occurring polymerases. Such variants typically will have substantial sequence identity or similarity to the wild-type or naturally occurring polymerase, typically at least 80% sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the unmodified DNA polymerase has reverse transcriptase (RT) activity and/or the ability to incorporate ribonucleotides or other T- modified nucleotides.
  • RT reverse transcriptase
  • Suitable polymerases also include, for example, certain chimeric DNA polymerases comprising polypeptide regions from two or more enzymes. Examples of such chimeric DNA polymerases are described in, e.g., U.S. Patent No. 6,228,628. Particularly suitable are chimeric CS-family DNA polymerases, which include the CS5 (SEQ ID NO: 18) and CS6 (SEQ ID NO: 19) polymerases and variants thereof having substantial sequence identity or similarity to SEQ ID NO: 18 or SEQ ID NO: 19 (typically at least 80% sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity).
  • the CS5 and CS6 DNA polymerases are chimeric enzymes derived from Thermus sp. Z05 and Thermotoga maritima (Tm ⁇ ) DNA polymerases. They comprise the N-terminal 5'-nuclease domain of the Thermus enzyme and the C-terminal 3'-5' exonuclease and the polymerase domains of the Tma enzyme. These enzymes have efficient reverse transcriptase activity, can extend nucleotide analog-containing primers, and can incorporate alpha-phosphorothioate dNTPs, dUTP, dITP, and also fluorescein- and cyanine-dye family labeled dNTPs.
  • the CS5 and CS6 polymerases are also efficient Mg 2+ -activated PCR enzymes. Nucleic acid sequences encoding CS5 and CS6 polymerases are provided in Figures 2B and 3B, respectively. CS5 and CS6 chimeric polymerases are further described in, e.g., U.S. Pat. Application Publication No. 2004/0005599.
  • the unmodified form of the DNA polymerase is a polymerase that has been previously modified, typically by recombinant means, to confer some selective advantage.
  • modifications include, for example, the amino acid substitutions G46E, L329A, and/or E678G in CS5 DNA polymerase, CS6 DNA polymerase, or corresponding mutation(s) in other polymerases.
  • the unmodified form of the DNA polymerase is one of the following (each having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19 except for the designated substitution(s)): G46E; G46E L329A; G46E E678G; or G46E L329A E678G.
  • the E678G substitution allows for the incorporation of ribonucleotides and other 2'-modified nucleotides, but this mutation also appears to result in an impaired ability to extend primed templates.
  • the mutations according to the present invention which result in a faster extension rate of the mutant polymerase, ameliorate this particular feature of the E678G mutation.
  • the mutant DNA polymerases of the present invention comprise one or more amino acid substitutions in the active site relative to the unmodified polymerase.
  • the amino acid substitution(s) are at at least one of the following amino acid positions: position X a8 of the motif set forth in SEQ ID NO:1; position X b8 of the motif set forth in SEQ ID NO:2; position X 04 of the motif set forth in SEQ ID NO: 3; and position X 06 of the motif set forth in SEQ ID NO:3.
  • amino acid substitution at one or more of these positions confers improved nucleotide- incorporating activity, yielding a mutant DNA polymerase with an improved (faster) nucleic acid extension rate relative to the unmodified polymerase.
  • amino acid substitution at one or more of these positions confers increased 3' - 5' exonuclease (proofreading) activity relative to the unmodified polymerase. While not intending to be limited to any particular theory, the present inventors believe that the improved nucleic acid extension rate of the mutant polymerases of the invention is a consequence of tighter binding to a template, i.e., less frequent dissociation from the template, resulting in a higher "processivity" enzyme.
  • mutant polymerase in, e.g., primer extension reactions relative to reactions involving the unmodified DNA polymerase.
  • the extension rate of the unmodified polymerase i.e., lacking the specific mutations that are the subject of the invention
  • the mutant polymerases also appear to perform much better than the unmodified forms at high ionic strength.
  • the performance of the unmodified polymerase at low ionic strength would approach that of the mutant polymerase.
  • the amino acid substitutions are single amino acid substitutions.
  • the mutant polymerase can, e.g., comprise any one of the amino acid substitutions at position X a8 , Xb 8 , X c 4, or X 06 separately.
  • the mutant polymerase comprises any one of various combinations of substitutions at two, three, or all four of these positions.
  • the mutant DNA polymerase of the invention comprises amino acid substitutions at each of positions X b8 and Xc 6 .
  • the amino acid at position X a8 , X b8 , X 04 , or X 06 is substituted with an amino acid that does not correspond to the respective motif as set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • the amino acid at position X a8 if substituted, is not Q, T, M, G or L; the amino acid at position X b8 , if substituted, is not D, E or N; the amino acid at position X 04 , if substituted, is not I or L; and/or the amino acid at position X 06 , if substituted, is not S, A, V or G.
  • amino acid substitutions include Arginine (R) at position X a8 , Glycine (G) at position X b8 , Phenylalanine (F) at position X 04 , and/or Phenylalanine (F) at position X 06 .
  • Other suitable amino acid substitution(s) at one or more of the identified sites can be determined using, e.g., known methods of site-directed mutagenesis and determination of primer extension performance in assays described further herein or otherwise known to persons of skill in the art.
  • the mutant DNA polymerase of the present invention is derived from CS5 DNA polymerase (SEQ ID NO: 18), CS6 DNA polymerase (SEQ ID NO: 19), or a variant of those polymerases (e.g. , G46E; G46E
  • the mutant polymerase comprises at least one amino acid substitution, relative to a CS5 DNA polymerase or a CS6 DNA polymerase, at S671, D640, Q601, and/or 1669.
  • Exemplary CS5 DNA polymerase and CS6 DNA polymerase mutants include those comprising the amino acid substitution(s) S671F, D640G, Q601R, and/or I669F.
  • the mutant CS5 polymerase or mutant CS6 polymerase comprises, e.g. , amino acid substitutions at both D640 and S671 (e.g., D640G and S671F).
  • Other, exemplary CS5 DNA polymerase and CS6 DNA polymerase mutants include the following (each having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19 except for the designated substitutions):
  • the mutant DNA polymerases of the present invention can also include other, non- substitutional modification(s). Such modifications can include, for example, covalent modifications known in the art to confer an additional advantage in applications comprising primer extension.
  • the mutant DNA polymerase further includes a thermally reversible covalent modification.
  • a modifier group is covalently attached to the protein, resulting in a loss of all, or nearly all, of the enzyme activity. The modifier group is chosen so that the modification is reversed by incubation at an elevated temperature.
  • DNA polymerases comprising such thermally reversible modifications are particularly suitable for hot-start applications, such as, e.g., various hot-start PCR techniques.
  • Thermally reversible modifier reagents amenable to use in accordance with the mutant DNA polymerases of the present invention are described in, for example, U.S. Patent No. 5,773,258 to Birch et al..
  • Exemplary modifications include, e.g. , reversible blocking of lysine residues by chemical modification of the ⁇ -amino group of lysine residues ⁇ see Birch et al, supra).
  • the thermally reversible covalent modification includes covalent attachment, to the ⁇ -amino group of lysine residues, of a dicarboxylic anhydride as described in Birch et al. , supra.
  • mutant polymerases comprising a thermally reversible covalent modification are produced by a reaction, carried out at alkaline pH at a temperature which is less than about 25°C, of a mixture of a thermostable enzyme and a dicarboxylic acid anhydride having a general formula as set forth in the following formula I:
  • R 1 and R 2 are hydrogen or organic radicals, which may be linked; or having the following formula II:
  • Ri and R 2 are organic radicals, which may linked, and the hydrogens are cis, essentially as described in Birch et al, supra.
  • the unmodified form of the polymerase is G64E CS5 DNA polymerase.
  • the mutant DNA polymerases of the present invention can be constructed by mutating the DNA sequences that encode the corresponding unmodified polymerase ⁇ e.g., a wild- type polymerase or a corresponding variant from which the mutant polymerase of the invention is derived), such as by using techniques commonly referred to as site-directed mutagenesis.
  • Nucleic acid molecules encoding the unmodified form of the polymerase can be mutated by a variety of polymerase chain reaction (PCR) techniques well-known to one of ordinary skill in the art. (See, e.g., PCR Strategies (M. A. Innis, D. H. Gelfand, and J. J.
  • the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site- directed mutants into a polynucleotide encoding an unmodified form of the polymerase.
  • two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site- directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site.
  • Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli.
  • Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli.
  • This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids.
  • the tight linkage of the two mutations and the subsequent linearization of unmutated plasmids result in high mutation efficiency and allow minimal screening.
  • this method requires the use of only one new primer type per mutation site.
  • a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously.
  • Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis, such as for example, on a Mutation Detection Enhancement gel (Mallinckrodt Baker, Inc., Phillipsburg, NJ) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control). Alternatively, the entire DNA region can be sequenced to confirm that no additional mutational events have occurred outside of the targeted region.
  • Verified mutant duplexes in pET (or other) overexpression vectors can be employed to transform E. coli such as, e.g., strain E. coli BL21 (DE3) pLysS, for high level production of the mutant protein, and purification by standard protocols.
  • the method of FAB-MS mapping for example, can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutagenized protein).
  • the set of cleavage fragments is fractionated by, for example, microbore HPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by standard methods, such as FAB-MS.
  • the determined mass of each fragment are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS data agrees with prediction.
  • CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide can be purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification.
  • Mutant DNA polymerases with more than one amino acid substituted can be generated in various ways. In the case of amino acids located close together in the polypeptide chain (as with amino acids X 04 and X 06 of the motif set forth in SEQ ID NO: 3), they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted.
  • the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
  • An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: DNA encoding the unmodified polymerase is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations.
  • the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
  • the multi-site mutagenesis method of Seyfang & Jin (Anal. Biochem. 324:285-291. 2004) may be utilized.
  • nucleic acids encoding any of the mutant DNA polymerases of the present invention.
  • a nucleic acid of the present invention encoding a mutant DNA polymerase
  • Any vector containing replicon and control sequences that are derived from a species compatible with the host cell can be used in the practice of the invention.
  • expression vectors include transcriptional and translational regulatory nucleic acid regions operably linked to the nucleic acid encoding the mutant DNA polymerase.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • the vector may contain a Positive Retroregulatory Element (PRE) to enhance the half-life of the transcribed mRNA (see Gelfand et al. U.S. Patent No. 4,666,848).
  • PRE Positive Retroregulatory Element
  • the transcriptional and translational regulatory nucleic acid regions will generally be appropriate to the host cell used to express the polymerase. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Vectors also typically include a polylinker region containing several restriction sites for insertion of foreign DNA.
  • "fusion flags" are used to facilitate purification and, if desired, subsequent removal of tag/flag sequence, e.g., "His-Tag". However, these are generally unnecessary when purifying an thermoactive and/or thermostable protein from a mesophilic host (e.g., E.
  • a "heat-step” where a "heat-step” may be employed.
  • suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes, and the mutant polymerase of interest are prepared using standard recombinant DNA procedures. Isolated plasmids, viral vectors, and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well-known in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, NY, 2nd ed. 1989)).
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes can include, for example, genes coding for ampicillin and/or tetracycline resistance, which enables cells transformed with these vectors to grow in the presence of these antibiotics.
  • a nucleic acid encoding a mutant DNA polymerase is introduced into a cell, either alone or in combination with a vector.
  • introduction into or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent integration, amplification, and/or expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO 4 precipitation, liposome fusion, LIPOFECTIN®, electroporation, viral infection, and the like.
  • Prokaryotes are typically used as host cells for the initial cloning steps of the present invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated.
  • Suitable prokaryotic host cells include E. coli Kl 2 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli Kl 2 strain DGl 16 (ATCC No. 53,606), E. coli Xl 776 (ATCC No. 31 ,537), and E. coli B; however many other strains of E.
  • coli such as HBlOl, JMlOl, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species can all be used as hosts.
  • Prokaryotic host cells or other host cells with rigid cell walls are typically transformed using the calcium chloride method as described in section 1.82 of Sambrook et al. , supra. Alternatively, electroporation can be used for transformation of these cells.
  • Prokaryote transformation techniques are set forth in, for example Dower, in Genetic Engineering, Principles and Methods 12:275- 296 (Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol, 204:63, 1991.
  • Plasmids typically used for transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUCl 19, and Bluescript Ml 3, all of which are described in sections 1.12-1.20 of Sambrook et al. , supra. However, many other suitable vectors are available as well.
  • the mutant DNA polymerases of the present invention are typically produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the mutant DNA polymerase, under the appropriate conditions to induce or cause expression of the mutant DNA polymerase.
  • Methods of culturing transformed host cells under conditions suitable for protein expression are well-known in the art (see, e.g., Sambrook et al., supra).
  • Suitable host cells for production of the mutant polymerases from lambda pL promotor-containing plasmid vectors include E. coli strain DGl 16 (ATCC No. 53606) ⁇ see US Pat. No. 5,079,352 and Lawyer, F.C. et al, PCR Methods and Applications 2:275-87, 1993).
  • the mutant polymerase can be harvested and isolated. Methods for purifying the thermostable DNA polymerase are described in, for example, Lawyer et al. , supra.
  • the ability of the mutant DNA polymerases to extend primed templates can be tested in any of various known assays for measuring nucleotide incorporation.
  • primed template molecules e.g., Ml 3 DNA, etc.
  • an appropriate buffer e.g., a complete set of dNTPs (e.g., dATP, dCTP, dGTP, and dTTP), and metal ion
  • DNA polymerases will extend the primers, converting single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA). This conversion can be detected and quantified by, e.g., adding a dsDNA-binding dye, such as SYBR Green I.
  • a dsDNA-binding dye such as SYBR Green I.
  • reaction plates can be taken (e.g., at 10-30 second intervals), thereby allowing the progress of the reactions to be followed.
  • the amount of fluorescence detected can be readily converted to extension rates.
  • extension rates of the mutants relative to the unmodified forms of polymerase can be determined.
  • the mutant DNA polymerases of the present invention may be used for any purpose in which such enzyme activity is necessary or desired. Accordingly, in another aspect of the invention, methods of primer extension using the mutant polymerases are provided. Conditions suitable for primer extension are known in the art. (See, e.g., Sambrook et al., supra. See also Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons 1999). Generally, a primer is annealed, i.e., hybridized, to a target nucleic acid to form a primer-template complex.
  • the primer-template complex is contacted with the mutant DNA polymerase and free nucleotides in a suitable environment to permit the addition of one or more nucleotides to the 3' end of the primer, thereby producing an extended primer complementary to the target nucleic acid.
  • the primer can include, e.g. , one or more nucleotide analog(s).
  • the free nucleotides can be conventional nucleotides, unconventional nucleotides (e.g., ribonucleotides or labeled nucleotides), or a mixture thereof.
  • the primer extension reaction comprises amplification of a target nucleic acid.
  • the primer extension reaction comprises reverse transcription of an RNA template (e.g., RT-PCR).
  • RNA template e.g., RT-PCR.
  • the mutant polymerases are used for primer extension in the context of DNA sequencing, DNA labeling, or labeling of primer extension products.
  • DNA sequencing by the Sanger dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. ScL USA 74: 5463, 1977) is improved by the present invention for polymerases capable of incorporating unconventional, chain-terminating nucleotides.
  • Advances in the basic Sanger et al. method have provided novel vectors (Yanisch- Perron et al., Gene 33:103-119, 1985) and base analogues (Mills et al, Proc. Natl. Acad.
  • DNA sequencing requires template-dependent primer extension in the presence of chain-terminating base analogs, resulting in a distribution of partial fragments that are subsequently separated by size.
  • the basic dideoxy sequencing procedure involves (i) annealing an oligonucleotide primer, optionally labeled, to a template; (ii) extending the primer with DNA polymerase in four separate reactions, each containing a mixture of unlabeled dNTPs and a limiting amount of one chain terminating agent such as a ddNTP, optionally labeled; and (iii) resolving the four sets of reaction products on a high-resolution denaturing polyacrylamide/urea gel.
  • the reaction products can be detected in the gel by autoradiography or by fluorescence detection, depending on the label used, and the image can be examined to infer the nucleotide sequence.
  • These methods utilize DNA polymerase such as the Klenow fragment of E. coli Pol I or a modified T7 DNA polymerase.
  • thermostable polymerases such as Taq DNA polymerase
  • cycle sequencing modification thereof referred to as "cycle sequencing”
  • mutant thermostable polymerases of the present invention can be used in conjunction with such methods.
  • cycle sequencing is a linear, asymmetric amplification of target sequences complementary to the template sequence in the presence of chain terminators. A single cycle produces a family of extension products of all possible lengths.
  • Cycle sequencing requires less template DNA than conventional chain-termination sequencing.
  • Thermostable DNA polymerases have several advantages in cycle sequencing; they tolerate the stringent annealing temperatures which are required for specific hybridization of primer to nucleic acid targets as well as tolerating the multiple cycles of high temperature denaturation which occur in each cycle, e.g., 90-95 0 C.
  • AMPLITAQ® DNA Polymerase and its derivatives and descendants e.g., AmpliTaq CS DNA Polymerase and AmpliTaq FS DNA Polymerase have been included in Taq cycle sequencing kits commercialized by companies such as Perkin- Elmer (Norwalk, CT) and Applied Biosystems (Foster City, CA).
  • Variations of chain termination sequencing methods include dye-primer sequencing and dye-terminator sequencing.
  • dye-primer sequencing the ddNTP terminators are unlabeled, and a labeled primer is utilized to detect extension products (Smith et al. , Nature 32:674-679, 1986).
  • dye-terminator DNA sequencing a DNA polymerase is used to incorporate dNTPs and fluorescently labeled ddNTPs onto the end of a DNA primer (Lee et al, Nuc. Acids. Res. 20:2471, 1992). This process offers the advantage of not having to synthesize dye labeled primers.
  • dye-terminator reactions are more convenient in that all four reactions can be performed in the same tube.
  • Both dye-primer and dye-terminator methods may be automated using an automated sequencing instrument produced by Applied Biosystems (Foster City, CA) (U.S. Pat. No. 5,171,534).
  • the completed sequencing reaction mixture is fractionated on a denaturing polyacrylamide gel or capillaries mounted in the instrument.
  • a laser at the bottom of the instrument detects the fluorescent products as they are electrophoresed according to size through the gel.
  • Two types of fluorescent dyes are commonly used to label the terminators used for dye- terminator sequencing-negatively charged and zwitterionic fluorescent dyes.
  • Negatively charged fluorescent dyes include those of the fluorescein and BODIPY families.
  • BODIPY dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are described in International Patent Publication WO 97/00967.
  • Zwitterionic fluorescent dyes include those of the rhodamine family.
  • Commercially available cycle sequencing kits use terminators labeled with rhodamine derivatives. However, the rhodamine-labeled terminators are rather costly and the product must be separated from unincorporated dye-ddNTPs before loading on the gel since they co-migrate with the sequencing products.
  • Rhodamine dye family terminators seem to stabilize hairpin structures in GC- rich regions, which causes the products to migrate anomalously. This requires the use of dITP, which relaxes the secondary structure but also affects the efficiency of incorporation of terminator.
  • fluorescein-labeled terminators eliminate the separation step prior to gel loading since they have a greater net negative charge and migrate faster than the sequencing products.
  • fluorescein-labeled sequencing products have better electrophoretic migration than sequencing products labeled with rhodamine.
  • wild-type Taq DNA polymerase does not efficiently incorporate terminators labeled with fluorescein family dyes, this can now be accomplished efficiently by use of the modified enzymes as described in U.S. Patent Application Publication No. 2002/0142333.
  • the unmodified DNA polymerase in accordance with the present invention is a modified thermostable polymerase as described in US 2002/0142333 and having the motifs set forth in SEQ ID NOs. 1, 2, and 3.
  • mutant DNA polymerases of the invention include those involving terminator compounds that include 2'-PO 4 analogs of ribonucleotides ⁇ see, e.g., U. S. Application Publication Nos. 2005/0037991 and 2005/0037398, and International Application No. WO 2005/026184, entitled "SYNTHESIS AND COMPOSITIONS OF NUCLEIC ACIDS COMPRISING 2'-TERMINATOR NUCLEOSIDES", filed June 29, 2004 by Bodepudi et al. and International Application No.
  • WO 2005/005667 entitled “2'-TERMINATOR RELATED PYROPHOSPHOROLYSIS ACTIVATED POLYMERIZATION", filed June 29, 2004 by Gelfand et al.).
  • the mutant DNA polymerases described herein generally improve these sequencing methods, e.g., by reducing the time necessary for the cycled extension reactions and/or by reducing the amount or concentration of enzyme that is utilized for satisfactory performance.
  • kits are provided for use in primer extension methods described herein.
  • the kit is compartmentalized for ease of use and contains at least one container providing a mutant DNA polymerase in accordance with the present invention.
  • One or more additional containers providing additional reagent(s) can also be included.
  • Such additional containers can include any reagents or other elements recognized by the skilled artisan for use in primer extension procedures in accordance with the methods described above, including reagents for use in, e.g., nucleic acid amplification procedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures.
  • the kit further includes a container providing a 5' sense primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template, or a primer pair comprising the 5' sense primer and a corresponding 3' antisense primer.
  • the kit includes one or more containers providing free nucleotides (conventional and/or unconventional).
  • the kit includes alpha-phophorothioate dNTPs, dUTP, dITP, and/or labeled dNTPs such as, e.g., fluorescein- or cyanin-dye family dNTPs.
  • the kit includes one or more containers providing a buffer suitable for a primer extension reaction.
  • Mutations in CS family polymerases were identified that provide, e.g., improved ability to extend primed DNA templates in the presence of free nucleotides.
  • the steps in this screening process included library generation, expression and partial purification of the mutant enzymes, screening of the enzymes for the desired property, DNA sequencing, clonal purification, and further characterization of selected candidate mutants, and generation, purification, and characterization of combinations of the mutations from the selected mutants. Each of these steps is described further below.
  • the mutations identified by this process include S671F, D640G, Q601R, and I669F, either separately or in various combinations.
  • mutant polymerases were placed in several CS-family polymerases, including G46E CS5, G46E L329A CS5, G46E E678G CS5, and G46E L329A E678G CS5. Some of these mutant polymerases are listed in Table 2. Other exemplary mutant polymerases that have been made include CS6 G46E Q601R D640G S671F E678G DNA polymerase and certain Thermus sp. Z05 DNA polymerase mutants. The resulting mutant polymerases were characterized by analyzing their performance in a series of Kinetic Thermal Cycling (KTC) experiments.
  • KTC Kinetic Thermal Cycling
  • the identified mutations, S671F, D640G, Q601R, and I669F resulted in, e.g., an improved ability to extend primed templates.
  • the S671F, D640G, Q601R, and I669F mutations ameliorated this property of impaired primer extension ability.
  • the identified mutations, particularly S671F alone and S671F plus D640G also showed improved efficiency of reverse transcription when placed in G46E CS5 and G46E L329A CS5 DNA polymerases. Additional features of the mutant DNA polymerases of the invention are described further below.
  • a nucleic acid encoding the polymerase domain of CS5 E678G DNA polymerase was subjected to error-prone (mutagenic) PCR between BgI II and Hind III restriction sites of a plasmid including this nucleic acid sequence.
  • PCR was performed using a range of Mg +2 concentrations from 1.8-3.5 mM, in order to generate libraries with a corresponding range of mutation rates.
  • Buffer conditions were: 50 mM Bicine pH 8.2, 115 mM KOAc, 8% w/v glycerol, 0.2 mM each dNTPs, and 0.2X SYBR Green I.
  • a GeneAmp® AccuRT Hot Start PCR enzyme was used at 0.15 U/ ⁇ l.
  • the resulting amplicon was purified over a Qiaquick spin column (Qiagen, Inc., Valencia, CA, USA) and cut with BgI II and Hind III, then re-purified,
  • CIP calf intestinal phosphatase
  • the cut vector and the mutated insert were mixed at different ratios and treated with T4 ligase overnight at 15 0 C.
  • the ligations were purified and transformed into an E.coli host strain by electroporation.
  • Extract library preparation Part 1 Fermentation: From the clonal libraries described above, a corresponding library of partially purified extracts suitable for screening purposes was prepared. The first step of this process was to make small-scale expression cultures of each clone. These cultures were grown in 96-well format; therefore there were 4 expression culture plates for each 384-well library plate. One ⁇ l was transferred from each well of the clonal library plate to a well of a 96 well seed plate, containing 150 ⁇ l of Medium A (see Table 3 below). This seed plate was shaken overnight at 1150 rpm at 3O 0 C, in an iEMS plate incubater/shaker (ThermoElectron).
  • Extract library preparation Part 2 ⁇ Extraction Cell pellets from the fermentation step were resuspended in 30 ⁇ l Lysis buffer (Table 4 below) and transferred to 384-well thermocycler plates. Note that the buffer contained lysozyme to assist in cell lysis, and two nucleases to remove both RNA and DNA from the extract. The plates were subjected to three rounds of freeze-thaw (-70 0 C freeze, 37 0 C thaw, not less than 15 minutes per step) to lyse the cells.
  • Ammonium sulfate was added (5 ⁇ l of a 0.75M solution) and the plates incubated at 75 0 C for 15 minutes in order to precipitate and inactivate contaminating proteins, including the exogenously added nucleases.
  • the plates were centrifuged at 3000 x g for 15 minutes and the supernatants transferred to a fresh 384-well thermocycler plate. These extract plates were frozen at -20 0 C for later use in screens. Each well contained about 0.5-3 ⁇ M of the mutant library polymerase enzyme.
  • Ml 3mpl 8 single-stranded DNA (Ml 3; GenBank Accession No. X02513), primed with an oligonucleotide having the following sequence:
  • Master mixes invariably included metal ion, usually magnesium at 1-4 mM, a mixture of all four dNTPs or dNTP analogs, buffer components to control the pH and the ionic strength, typically 25 mM Tricine pH 8.3/35 mM KOAc, and SYBR Green I at 0.6X (Molecular Probes), which allowed for the fluorescent detection of primer strand extension.
  • metal ion usually magnesium at 1-4 mM
  • a mixture of all four dNTPs or dNTP analogs buffer components to control the pH and the ionic strength, typically 25 mM Tricine pH 8.3/35 mM KOAc, and SYBR Green I at 0.6X (Molecular Probes), which allowed for the fluorescent detection of primer strand extension.
  • a metal chelator such as EDTA
  • extension reactions were run in the presence and absence of ribonucleotides and the resulting rates of extension were compared, using the methods described above.
  • Adding a high level of ribonucleotide for example, a 50:50 mix of rATP and dATP
  • G46E L329A E678G CS5 reduced the rate of extension of the parental enzyme, G46E L329A E678G CS5.
  • Mutant extracts that exhibited a reduced level of inhibition by ribonucleotides were identified in this screen. Primary screening was done on the scale of thousands of extracts. The top several percent of these were chosen for re-screening.
  • the double combination mutants tested including D640G S671F and Q601R S671F, also showed improved extension rates relative to strains carrying only a single mutation. Moreover, the combination mutants also demonstrated improved rates of extension on primed Ml 3 DNA when only dNTPs were present, when compared to the parental type, and furthermore it was observed that the degree of improvement relative to the parental type was greatest when the extension rate experiment was performed at low enzyme concentration or relatively high salt concentration. These observations were repeated when the combination mutations were moved into a genetic backbone that did not include the riboincorporating mutation E678G.
  • the nucleic acid extension rates of various mutants of G46E L329A E678G CS5 DNA polymerase were determined in the presence of 90% riboadenosine triphosphate (ribo ATP or rATP).
  • the reaction mixture contained 25 mM Tricine pH 8.3, 20 mM ( Figure 4) or 60 mM ⁇ Figure 5) KOAc, 3 mM MgCl 2 , 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, IX SYBR Green I, 0.5 nM primed M13, and 5 nM enzyme.
  • nucleotides were added to a final concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, 0.01 mM dATP, and 0.09 mM ribo ATP.
  • Parallel reactions containing no nucleotides were also set up. All reactions were run in quadruplicate in 20 ⁇ l volume in 384 well thermocycler plates. The extension of primed Ml 3 template was monitored by fluorescence in a kinetic thermocycler set at 64 0 C, taking readings every 10 seconds. Replicate identical reactions were averaged and the parallel minus nucleotide reactions subtracted. Extension rate was estimated by linear regression analysis of the resulting data.
  • Figures 4 and 5 show results obtained from these analyses.
  • Figures 4 and 5 illustrate that improved nucleic acid extension rates result from various mutants described herein, when ribonucleotides are present in reaction mixtures and incorporated on a DNA template.
  • Figures 4 and 5 illustrate that improved nucleic acid extension rates result from various mutants described herein, when ribonucleotides are present in reaction mixtures and incorporated on a DNA template.
  • certain mutations are combined in a single mutant enzyme, even further extension rate improvements are observed.
  • the nucleic acid extension rate of various mutants of G46E L329A CS5 DNA polymerase, as well as Thermus sp. Z05 DNA polymerase and its truncate, delta Z05 DNA polymerase was determined.
  • the reaction mixture contained 25 mM Tricine pH 8.3, 0 mM ( Figure 6) or 60 mM ⁇ Figure T) KOAc, 3 mM MgCl 2 , 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, IX SYBR Green I, 0.5 nM primed Ml 3, and 5 nM enzyme.
  • nucleotides were added to a final concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, and 0.1 mM dATP.
  • Parallel reactions containing no nucleotides were also set up. All reactions were run in quadruplicate in 20 ⁇ l volume in 384 well thermocycler plates. The extension of primed Ml 3 template was monitored by fluorescence in a kinetic thermocycler set at 64 0 C, taking readings every 10 seconds. Replicate identical reactions were averaged and the parallel minus nucleotide reactions subtracted. Extension rate was estimated by linear regression analysis of the resulting data.
  • FIG. 6 and 7 illustrate, e.g., that certain mutations described herein result in improved nucleic acid extension rates even when ribonucleotides are not present in the reaction mixtures, and even in a genetic backbone that does not include the ribonucleotide-incorporation mutation, E678G. As further shown, for example, this rate improvement is even greater when the mutations are combined in a single mutant enzyme.
  • the reaction mixture contained 25 mM Tricine pH 8.3, 0-100 mM KOAc, 3 mM MgCl 2 , 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, IX SYBR Green I, 0.5 nM primed M13, and 25 nM ( Figure 8) or 5 nM ( Figure 9) enzyme.
  • nucleotides were added to a final concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, and 0.1 mM dATP.
  • Parallel reactions containing no nucleotides were also set up. All reactions were run in quadruplicate in 20 ⁇ l volume in 384 well thermocycler plates. The extension of primed Ml 3 template was monitored by fluorescence in a kinetic thermocycler set at 64 0 C, taking readings every 10 seconds. Replicate identical reactions were averaged and the parallel minus nucleotide reactions subtracted. Extension rate was estimated by linear regression analysis of the resulting data.
  • EXAMPLE V USE OF VARIOUS MUTANT CS5 DNA POLYMERASES IN RT-PCR
  • Mg 2+ -based RT The mutations Q601R, D640G, and S671F, separately and in combination, were evaluated for their effect on PCR and RT-PCR efficiency in the presence OfMg +2 .
  • the reactions all contained the following components: 50 mM Tricine pH 8.0, 2.5 mM Mg(OAc) 2 , 6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 0.2X SYBR Green I, 0.02 units/ ⁇ l UNG, 0.2 mM each dATP, dCTP, and dGTP, 0.3 mM dUTP, and 0.03 mM dTTP, and 200 nM of each primer, wherein the primers comprise a 2'-amino- C at the 3 '-end.
  • Enzymes were used at their pre-determined concentration and KOAc optima. These are given in Table 5.
  • thermocycling parameters were: 50 0 C for 2 minutes; 65 0 C for 45 minutes; 93 0 C for 1 minute; then 40 cycles of: 93 0 C for 15 seconds; and 65 0 C for 30 seconds.
  • FIG. 10 Fluorescence data was analyzed to determine Ct values (emergence of fluorescence over baseline) ( Figure 10). More specifically, the data shown in Figure 10 (see also, Figure 11) illustrates, among other properties, e.g., that the mutations described herein, either singly or in combination, improve the efficiency of the Mg 2+ -activated reverse transcription activity of the mutant enzyme relative to the corresponding parent or non- mutant enzyme.
  • the GLDS enzyme performed well, e.g., when the time allowed for reverse transcription was descreased to 5 minutes, as shown in Figure 11 (referred to additionally below).
  • Mg 2+ -based RT with reduced RT time The mutations Q601R, D640G, and S671F, separately and in combination, were evaluated for their effect on RT-PCR efficiency in the presence of Mg +2 , using either 45 minute or 5 minute RT time.
  • the reactions all contained the following components: 50 mM Tricine pH 8.0, 2.5 mM Mg(OAc) 2 , 6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 1% DMSO, 0.2X SYBR Green I, 0.02 units/ ⁇ l UNG, 0.2 mM each dATP, dCTP, and dGTP, 0.3 mM dUTP, and 0.03 mM dTTP, and 200 nM of each primer, wherein the primers comprise a 2'-amino-C at the 3 '-end.
  • Enzymes were used at their pre-determined concentration and KOAc optima. These are given in the following Tables 6 and 7:
  • denotes condition that was not done
  • thermocycling parameters were: 50 0 C for 2 minutes; 65 0 C for 5 minutes or 45 minutes; 93 0 C for 1 minute; then 40 cycles of: 93 °C for 15 seconds; and 65 0 C for 30 seconds. Fluorescence data was analyzed to determine Ct values (emergence of fluorescence over baseline) ( Figure U).
  • Mn 2+ -based RT with reduced RT time The mutations Q601 R, D640G, and S671 F, separately and in combination, were evaluated for their effect on RT-PCR efficiency in the presence of Mn +2 , using either 45 minute or 5 minute RT time.
  • the reactions all contained the following components: 50 mM Tricine pH 8.0, 1 mM Mn(OAc) 2 , 6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 1 % DMSO, 0.2X SYBR Green I, 0.02 units/ ⁇ l UNG, 0.2 mM each dATP, dCTP, and dGTP, 0.3 mM dUTP, and 0.03 mM dTTP, and 200 nM of each primer, wherein the primers comprise a 2'-amino-C at the 3 '-end.
  • Enzymes were used at their pre-determined concentration/KOAc optima. These are given in the following Tables 8 and 9: Table 8. 45 minute RT Time:
  • denotes condition that was not done
  • thermocycling parameters were: 50 0 C for 2 minutes; 65 0 C for 5 minutes or 45 minutes; 93 0 C for 1 minute; then 40 cycles of: 93 0 C for 15 seconds; and 65 0 C for 30 seconds. Fluorescence data was analyzed to determine Ct values (emergence of fluorescence over baseline) ( Figure 12).
  • the data shown in Figure 12 illustrates, among other properties, e.g., that improved Mn 2+ -activated reverse transcription efficiency results from certain of the mutations described herein, either singly or in combination, and that this improvement is enhanced when the time allowed for reverse transcription is decreased.
  • RNA polymerases it is sometimes useful to fragment a PCR product, for example when analyzing the product in a hybridization-based assay. Fragmentation can be easily accomplished by treating with alkali and heat, if ribonucleotides have been incorporated into the PCR product. For such applications relatively low level ribo-substitution will suffice to achieve fragments of optimal length.
  • the ability of various mutant DNA polymerases to generate ribo-substituted PCR product of length 1 kb was demonstrated in the following example.
  • the reaction mixture was composed of 100 mM Tricine pH 8.3, 75 mM KOAc, 5% v/v glycerol, 2.5 mM Mg(OAc) 2 , 50 nM enzyme, 0.1% v/v DMSO, and 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20).
  • Various mixtures of dNTPs and rNTPS were tested. In all cases, the sum of rATP and dATP was 200 ⁇ M, as was the sum of dCTP and rCTP, and dGTP and rGTP.
  • dTTP and rTTP were 40 ⁇ M and the sum of dUTP and rUTP was 360 ⁇ M.
  • rNTP Series in Figures 13 A and B (% rNTP indicated above the relevant lane in the gel)
  • rATP alone was added, up to 50% of the total (see,
  • rATP Series in Figures 13 A and B (% rATP indicated above the relevant lane in the gel)).
  • This reaction mix included primers used to generate a 1 kb product from an Ml 3 template.
  • the primers were used at 200 nM each, wherein the primers comprise a 2'- amino-C at the 3 '-end.
  • Ml 3 DNA was added to 10 6 copies per 100 ⁇ l reaction.
  • thermocycling parameters were: 50 0 C for 15 seconds; 92 0 C for 1 minute; then 30 cycles of: 92 0 C for 15 seconds; followed by an extension step of 62 0 C for 4 minutes.
  • the ability to make full length amplicon under the various conditions tested was determined by agarose gel electrophoresis, loading 5 ⁇ l of each reaction per lane on a 2% egel-48 (Invitrogen) ( Figures J3A and J 3B).
  • these figures show, e.g., that certain mutant enzymes described herein are able to produce full-length (1 kb) amplicons at higher levels of ribonucleotides present in the reaction mixtures than the corresponding parental or non-mutant G46E CS5R enzyme.
  • the mixture of GL CS5 and GLE enzymes made amplicon at the highest level of ribonucleotide assayed in this example, but because GL CS5 polymerase cannot incorporate ribonucleotides, these amplicons contained a relatively low level of ribonucleotides incorporated in the amplicon.
  • amplicons were then fragmented as follows: 2 ⁇ l amplicon was diluted 27.5X in 0.3N NaOH and 20 mM EDTA, then heated at 98 0 C for 10 minutes. The fragmented amplicon was neutralized by adding 2.5 ⁇ l 6 N HCl. To determine the degree of fragmentation achieved, the copy number of an internal fragment of the amplicon was compared before and after fragmentation, using quantitative PCR without UNG. The cycle delay observed due to fragmentation is an indication of the degree of fragmentation (and of ribonucleotide incorporation). Increased ribonucleotide incorporation leads to increased Ct delay. For this amplification, the reaction mixture was composed of 100 mM Tricine pH 8.3, 50 mM KOAc, 5% v/v glycerol, 2.5 mM
  • thermocycling parameters were: 50 0 C for 15 seconds; 92 0 C for 1 minute; then 46 cycles of: 92 0 C for 15 seconds; followed by an extension step of 62 0 C for 1 minute.
  • Threshold Cts were determined and corresponding fragmented and unfragmented Cts were compared, thus generating a delta Ct for each enzyme/rNTP condition tested.
  • the greater the amount of incorporated NTP reflecting an improved ability to incorporate NTPs in the presence of dNTPs
  • the data show, e.g., that the mutant enzymes of the invention are superior in the incorporation of ATP or NTP in generating PCR products that have increased extents of ribonucleotide substitution. Compare any of the illustrated enzymes to the parental blend "GL/GLE” or to "C5R". Increased fragmentation derives from increased ribonucleotide incorporation and an improved ability to incorporate a limiting concentration of ribonucleotides in the presence deoxynucleotides.
  • Hybridization assays frequently involve attaching biotin to the molecule being detected. It is therefore useful to incorporate biotin into PCR product. If biotin is attached to ribonucleotide, each fragment (except the 3' most distal fragment, which is usually complementary to the other primer and therefore uninformative) will carry a single biotin moiety, which will result in equal signal generation by each fragment.
  • the ability of various enzymes to incorporate ribo-nucleotides linked to a biotin into PCR product was determined, as described below.
  • the reaction mixture was composed of 100 raM Tricine pH 8.3, 75 mM KOAc, 5% v/v glycerol, 2.5 mM Mg(OAc) 2 , 50 nM enzyme, 0.1% DMSO, 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 200 ⁇ M each dCTP+ analogs, dGTP, and dATP, 360 ⁇ M dUTP, and 40 ⁇ M dTTP.
  • This reaction mix was used to generate a 1 kb product from an Ml 3 template, using primer sequences comprising a 2'-amino-C at 200 nM each.
  • Ml 3 DNA was added to 5 x 10 5 copies per 50 ⁇ l reaction.
  • Reactions were run in an ABI 9700 thermocycler. The thermocycling parameters were: 50 0 C for 15 seconds; 92 0 C for 1 minute; then 30 cycles of: 92 0 C for 15 seconds; followed by an extension step of 62 0 C for 4 minutes.
  • Figure 15A and B show, e.g., that mutants GQDSE and GDSE are both able to produce amplicon in higher levels of rCTP and biotinylated rCTP than can the corresponding parental or non-mutant G46E CS5R enzyme. Further while the GL/GLE blend can produce amplicon, this amplicon will have a low level of either rCTP or biotinylated rCTP incorporation, because the GL enzyme cannot incorporate these compounds.
  • amplicons were then fragmented as follows: 2 ⁇ l amplicon was diluted 27.5X in 0.3N NaOH and 20 mM EDTA, then heated at 98 0 C for 10 minutes. The fragmented amplicon was neutralized by adding 2.5 ⁇ l 6 N HCl. To determine the degree of fragmentation achieved, the copy number of an internal fragment of the amplicon was compared before and after fragmentation, using quantitative PCR without UNG. The cycle delay observed due to fragmentation is an indication of the degree of fragmentation (and of ribonucleotide incorporation). Thus, increased ribonucleotide incorporation leads to an increased Ct delay.
  • the reaction mixture was composed of 100 mM Tricine pH 8.3, 50 mM KOAc, 5% v/v glycerol, 2.5 mM Mg(OAc) 2 , 20 nM GQDS, 0.5% DMSO, 0.1 X SYBR Green I, 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 200 ⁇ M each dCTP, dGTP, and dATP, 360 ⁇ M dUTP, and 40 ⁇ M dTTP.
  • This reaction mix was used to generate a 340 bp product from the fragmented and unfragmented amplicons, diluting these templates a further 10,000-fold from the dilution used for fragmentation.
  • the primer sequences were used at 200 nM each, wherein each primer comprised a 2'-amino-C.
  • thermocycling parameters were: 50 0 C for 15 seconds; 92 0 C for 1 minute; then 46 cycles of: 92 0 C for 15 seconds; followed by an extension step of 62 0 C for 1 minute.
  • Threshold Cts were determined and corresponding fragmented and unfragmented Cts were compared, generating a delta Ct for each enzyme/rNTP condition tested. These are shown in Figures 16A and 16B.
  • Figures 16A and 16B illustrate, e.g., that an increase in the degree of fragmentation can be achieved by the mutant enzymes when using either rCTP or biotinylated rCTP, because they are able to produce amplicon with a higher level of ribonucleotide incorporation than the corresponding parental enzymes.
  • the reaction buffer was comprised of 100 mM Tricine pH 8.0, 2.5-50 mM G46E L329A E678G CS5 DNA polymerase or 2.5-50 mM G46E L329A D640G S671F E678G CS5 DNA polymerase, 50 nM KOAc, 10% v/v glycerol, 0.04 U/ ⁇ l UNG, 4 mM Mg(OAc) 2 , 1 % DMSO, 0.2X SYBR Green I, 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 0.2 mM each dATP, dCTP, and dGTP, and 0.4 mM dUTP, and 100 ⁇ M pyrophosphate.
  • Ml 3 template and enzyme were cross- titrated.
  • Ml 3 concentrations used were 0, 10 4 , 10 5 , and 10 6 copies per 20 ⁇ l reaction.
  • Enzyme concentrations used were 2.5 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 35 nM, and 50 nM.
  • Reactions were set up in triplicate in a 384-well thermocycler, using the following cycling parameters: 50 0 C for 2 minutes; 90 0 C for 1 minute; then 46 cycles of: 90 0 C for 15 seconds followed by an extension temperature of 62 0 C for 60 seconds.
  • One of the primers comprised a 2'-amino-C at the 3 '-end and the other primer comprised a 2'-PO 4 -A (i.e., a 2'-terminator nucleotide) at the 3'-end.
  • These primers added to the reaction mix at 0.1 ⁇ M each, will result in a 348 bp product from Ml 3 template.
  • the 2'-PO 4 -A residue at the 3' end of the second primer effectively acts as a terminator. In order to serve as a primer, it must be activated by pyrophosphorolytic removal of the terminal residue.
  • EXAMPLE VIII EFFECT QF SELECTED MUTATIONS ON THE EXTENSION RATE OF THERMUS SP. Z05 DNA POLYMERASE
  • the resulting amplicon includes the introduced mutation and also is designed to span vector-unique restriction sites, which can then be used to clone the amplicon into the vector plasmid DNA. Diagnostic restriction sites may also be introduced into the mutagenic primers as needed, in order to facilitate selection of the desired mutation from the resulting clones, which may include a mixture of mutants and wild-type clones. This procedure may introduce undesired mutations caused by low fidelity PCR, and hence it is necessary to sequence the resulting clones to confirm that only the desired mutations were created. Once the mutations were confirmed, they were combined with each other or with the previously isolated E683R mutation (ES 112) (see, U.S. Pat. Appl. No. 20020012970, entitled "High temperature reverse transcription using mutant DNA polymerases” filed March 30, 2001 by Smith et al.) by restriction fragment swaps, as described previously.
  • the reaction mixture contained 25 mM Tricine pH 8.3, 100 mM KOAc, 3 mM MgCl 2 , 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, IX SYBR Green I, 0.5 nM primed Ml 3, and 5 nM enzyme.
  • nucleotides were added to a final concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, and 0.1 mM dATP.
  • Parallel reactions containing no nucleotides were also set up. All reactions were run in quadruplicate in 20 ⁇ l volume in 384 well thermocycler plates. The extension of primed Ml 3 template was monitored by fluorescence in a kinetic thermocycler set at 64 0 C, taking readings every 10 seconds. Identical reactions were averaged and the parallel minus nucleotide reactions subtracted. Extension rate (see, Figure 19) was estimated by linear regression analysis of the resulting data. This data indicates, e.g., that in some cases the mutations described herein also have beneficial effects in the context of a non-chimeric Thermus DNA polymerase.
  • FIG. 20 is a photograph of a gel that shows the detection of the PCR products under the varied reaction conditions utilized in this analysis. This data illustrates, e.g., the improved amplification specificity and sensitivity that can be achieved using the blocked primers described herein relative to reactions not using those primers.
  • GLQDSE CS5 DNA polymerase refers to a G46E L329A Q601R D640G S671F E678G CS5 DNA polymerase.
  • Tth Storage Buffer included 0.2% Tween 20, 2OmM Tris pH8.0, 0.ImM EDTA, 10OmM KCl, ImM DTT, and 50% v/v glycerol. In addition, each reaction volume was brought to 50 ⁇ l with diethylpyrocarbonate (DEPC) treated water.
  • DEPC diethylpyrocarbonate
  • the varied reaction components included unblocked primers (see, the reactions denoted “unblocked primers” in Figure 20) and primers blocked with a 2'-Phosphate-U (i.e., a 2'-terminator nucleotide comprising a phosphate group at the 2' position, see, the reactions denoted "blocked primers” in Figure 20).
  • the reactions also either included (see, the reactions denoted “25ng Genomic DNA” in Figure 20) or lacked (see, the reactions denoted "Clean Target” in Figure 20) 25ng of human genomic DNA added to the mixtures.
  • the reactions also included 10 , 10 , 10 3 , 10 2 , or 10 1 copies of linearized plasmid DNA, which included the target nucleic acid, diluted in 1 ⁇ l HIV Specimen Diluent (10 mM Tris, 0.1 mM EDTA, 20 ⁇ g/mL Poly A, and 0.09% NaN 3 ) or 1 ⁇ l HIV Specimen Diluent in "Neg" reactions.
  • the indicated primer pairs amplified a 170 base pair product from the plasmid DNA.
  • Figure 21 is a graph that shows threshold cycle (C T ) values (y-axis) observed for the various mutant K-Ras plasmid template copy numbers (x-axis) utilized in these reactions.
  • Figure 21 further illustrates, e.g., the improved discrimination that can be achieved using the blocked primers described herein.
  • GDSE CS5 DNA polymerase refers to a G46E D640G S671F E678G CS5 DNA polymerase.
  • each reaction volume was brought to 50 ⁇ l with DEPC treated water.
  • the varied reaction components included the unblocked primers (see, the reactions denoted "unblocked” in Figure 21) and primers blocked with a 2'-Phosphate-C or a 2'- Phosphate-A (i.e., 2 '-terminator nucleotides comprising phosphate groups at 2' positions).
  • 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 or 0 copies (NTC reactions) (lOe ⁇ c, 10e5c, 10e4c, 10e3c, 10e2c, lOelc, and NTC, respectively, in Figure 21) of linearized mutant K-Ras plasmid DNA were added to the reactions.
  • mutant plasmid DNA was diluted in 1 ⁇ l HIV Specimen Diluent (see, above) or 1 ⁇ l HIV Specimen Diluent (see, above) in "NTC" reactions. Additionally, 10 6 copies of linearized wild-type K-Ras plasmid DNA were present in all reactions.
  • the wild-type K-Ras plasmid DNA was identical in sequence to mutant plasmid DNA except that it creates a C:C mismatch with the ultimate 3' base (dC) in primers 5 and 7.
  • Figure 22 is a graph that shows threshold cycle (C T ) values (y-axis) observed for the various enzymes and concentrations (x-axis) utilized in these reactions. These data show, e.g., the improved PAP amplification efficiencies that can be achieved using certain enzymes described herein.
  • the reaction components included primers blocked with a 2'-Phosphate-U or a 2'- Phosphate-A (i.e., 2 '-terminator nucleotides comprising phosphate groups at 2' positions).
  • the primer pairs created a 92 base pair amplicon on the linearized K-Ras plasmid template.
  • each reaction volume was brought to 50 ⁇ l with diethylpyrocarbonate (DEPC) treated water.
  • DEPC diethylpyrocarbonate
  • the polymerase concentration and KOAc concentrations were optimized for each individual polymerase as follows:
  • GLQDSE refers to a G46E L329A Q601R D640G S671F E678G CS5 DNA polymerase
  • GLE refers to a G46E E678G CS5 DNA polymerase
  • FIG. 23 is a graph that shows threshold cycle (Ct) values (y-axis) observed for the various enzymes (x-axis) utilized in these reactions in which the cDNA was measured using real-time PCR involving 5 '-nuclease probes.
  • Ct threshold cycle
  • the varied reaction components included a 3'-OH unblocked primer (see, the reactions denoted “3' OH Primer (Unblocked)" in Figure 23) and a primer blocked with a T- Phosphate-A or a 2'-monophosphate-3'-hydroxyl adenosine nucleotide (i.e., 2' terminator nucleotide comprising a phosphate group at the 2' position, see, the reactions denoted "2'P04 (Blocked)" in Figure 23). Further, the following polymerase conditions were compared in the cDNA reactions (see, Figure 23):
  • GLQDSE CS5 DNA polymerase refers to a G46E L329A Q601R D640G S671F E678G CS5 DNA polymerase and "GLQDS CS5 DNA polymerase” refers to a G46E L329A Q601R D640G S671F CS5 DNA polymerase.
  • each reaction was brought to 20 ⁇ l with diethylpyrocarbonate (DEPC) treated water.
  • DEPC diethylpyrocarbonate
  • RT reactions were incubated at 60 °C for 60 minutes in an ABI 9600 Thermal Cycler. After the RT incubation, RT reactions were diluted 100-fold in DEPC treated water. The presence of cDNA was confirmed and quantitated by 5 'nuclease probe- based real-time HCV PCR reactions designed to specifically measure the HCV cDNA products of the RT reactions. These reactions were performed using an ABI Prism 7700 Sequence Detector with the following temperature profile:
  • Figure 24 shows PCR growth curves of BRAF oncogene amplifications that were generated when bidirectional PAP was performed.
  • the x-axis shows normalized, accumulated fluorescence and the y-axis shows cycles of PAP PCR amplification. More specifically, these data were produced when mutation-specific amplification of the T ⁇ A mutation responsible for the V599E codon change in the BRAF oncogene (see, Brose et al. (2002) Cancer Res 62:6997-7000) was performed using 2 '-terminator blocked primers that overlap at their 3 '-terminal nucleotide at the precise position of the mutation. When primers specific to wild-type sequence were reacted to wild-type target or mutant target, only wild-type target was detected. Conversely, when primers specific to mutant sequence were reacted to wild-type target or mutant target, only mutant target was detected.
  • GLQDSE refers to a G46E L329A Q601R D640G S671F E678G CS5 DNA polymerase.
  • the varied reaction components included the wild-type BRAF primers blocked with a 2 '-Phosphate- A, a 2'-monophosphate-3'-hydroxyl adenosine nucleotide, a 2'- Phosphate-U or a 2'-monophosphate-3'-hydroxyl uridine nucleotide (i.e., 2' terminator nucleotides comprising a phosphate group at the 2' position, labeled "F5W/R5W" in Figure 24).
  • each reaction was brought to 50 ⁇ l with DEPC treated water.
  • Wild-type reactions (labeled “WT” in Figure 24) contained linearized DNA plasmid of the BRAF wild-type sequence and mutant reactions (labeled “MT” in Figure 24) contained linearized DNA plasmid of the BRAF mutant sequence.
  • Negative reactions (labeled “NEG” in Figure 24) contained HIV specimen diluent (10 raM Tris, 0.1 mM EDTA, 20 ⁇ g/mL Poly A, and 0.09% NaN 3 ) with no DNA. Combinations of the primers in PCR produced a 50 bp amplicon. Further, the reactions were performed using an ABI Prism 7700 Sequence Detector with the following temperature profile: 50 °C 1 minutes
  • This prophetic example illustrates a real-time monitoring protocol that involves PAP activation in which a blocked primer leads to the production of detectable signal as that primer is activated and extended.
  • the primer QX below is a DNA oligonucleotide that includes a quenching dye molecule, Black Hole Quencher® (BHQ) (Biosearch Technologies, Inc.) attached to the thirteenth nucleotide (A) from the 3' terminus.
  • BHQ Black Hole Quencher®
  • An oligonucleotide primer of the QX is mixed in solution with a complimentary oligonucleotide Rl (see, below) such that they form a hybrid duplex.
  • This duplex is further mixed with the reagents in the Table 10 provided below which notably include a fluorescein-labeled deoxyriboadenine tetraphosnhate (i.e.. a fluorescein-labeled T- terminator nucleotide) and DNA polymerase capable of incorporating such labeled tetraphosphate. See, U.S. Patent Application Nos.
  • the newly elongated Primer QX ⁇ FAM are purified from the mixture above using any number of purification methods known to persons of skill in the art.
  • An example of such a method capable of purifying Primer QX F from the mixture is High Peformance Liquid Chromatography (HPLC).
  • HPLC purification parameters are selected such that the preparation of Primer QX F ⁇ M is substantially free of non-extended Primer QX and fluorescein-labeled adenine tetraphosphates.
  • Dual HPLC Reverse Phase and Anion Exchange HPLC is known as a method for purifying such molecules.
  • molecules such as Primer QX FAM which contain a BHQ quenching molecule and a fluorescein molecule on the same oligonucleotide generally exhibit a suppressed fluorescein signal due to energy absorbance by the BHQ2 "quencher" molecule.
  • Primer QX FAM is synthesized chemically as described in, e.g., U.S. Patent Publication No. 2007/0219361.
  • each reaction is brought to 50 ⁇ l with DEPC treated water.
  • Some reactions contain a target sequence which serves as a substrate for PCR amplification, while others contain no target.
  • the target can be a DNA sequence identical to the 5'UTR region of the HCV genome. Combinations of these primers in PCR are expected to produce an approximately 244 bp amplicon.
  • the reactions can be performed using an ABI Prism 7700 Sequence Detector with the following temperature profile:
  • EXAMPLE XV EFFECT D580K, D580L, D580R AND D580T MUTATIONS ON THE EXTENSION RATE Z05 DNA POLYMERASE
  • the effect of various substitutions at the D580 position on the nucleic acid extension rate of Z05 DNA polymerase was determined.
  • the mutations were created in Z05 DNA polymerase, utilizing the technique of overlap PCR, and the mutant enzymes purified and quantified, as described previously.
  • the extension rate on primed M 13 (single-stranded DNA) template was determined, using both Mg +2 and Mn +2 as the metal co-factor, by monitoring the increase in SYBR Green I florescence, as described in Example II above, and elsewhere.
  • the reaction mixture contained 50 mM Tricine pH 8.3, 40 mM KOAc, 1 mM Mn(OAc) 2 or 2.5 mM Mg(OAc) 2 , 1.25% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, 0.6X SYBR Green I, 1.0 nM primed M13, and or 5 nM enzyme.
  • nucleotides were added to a final concentration of 0.2 mM dGTP, 0.2 mM dTTP, and 0.2 mM dCTP, and 0.2 mM dATP.
  • Parallel reactions containing no nucleotides were also set up. All reactions were run in quadruplicate in 20 ⁇ l volume in 384 well thermocycler plates. The extension of primed M13 template was monitored by fluorescence in a kinetic thermocycler set at 64 0 C, taking readings every 15 seconds. Replicate identical reactions were averaged and the parallel minus nucleotide reactions subtracted. Extension rate was estimated by linear regression analysis of the resulting data. Results are shown in Table 12 below:
  • Mn 2+ -based RT The mutations D580G, D580K, and D580R were evaluated for their effect on RT-PCR efficiency in the presence of Mn + .
  • the reactions all contained the following components: 55 mM Tricine pH 8.3, 4% v/v glycerol, 5% v/v DMSO, 110 mM KOAc, 2.7 mM Mn(OAc) 2 , 3.6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 0.04 units/ ⁇ l UNG, 0.45 mM each dATP, dCTP, dUTP, dGTP; 750 nM of each primer, wherein each primer comprised a t-butyl benzyl dA at the 3 '-end; and 150 nM of a Taq
  • Table 13 shows the Ct values obtained from the FAM signal increase due to cleavage of the TaqMan probe:
  • Mg 2+ -based RT The mutations D580G and D580K, were compared to ES 112 (Z05 E683R) for their ability to perform RT-PCR in the presence of Mg +2 .
  • the parental enzyme, Z05 DNA polymerase is known to perform Mg+ -based RT-PCR with greatly delayed Ct values relative to ESl 12, and was not re-tested in this study.
  • the conditions used were identical to those described immediately above, except that the KOAc was changed to 50 mM, the Mn(OAc) 2 was replaced with 2 mM Mg(OAC) 2 , and the enzyme concentration was reduced to 10 nM.
  • Thermocycling conditions were identical, except that only the 30 minute RT time was tested.
  • Table H shows the Ct values obtained from the FAM signal increase due to cleavage of the TaqMan probe: Table 14

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Abstract

L'invention porte sur des ADN polymérases mutantes possédant des vitesses d'extension améliorées par rapport à la polymérase non modifiée correspondante. Les polymérases mutantes précitées sont utilisées dans une variété de procédés d'extension d'amorce. L'invention concerne également des compositions associées, y compris des acides nucléiques recombinants, des vecteurs et des cellules hôtes qui sont utilisés, p.ex., dans la production des ADN polymérases mutantes.
EP07819100.4A 2006-10-18 2007-10-18 Adn polymérases mutantes et procédés associés Active EP2079834B1 (fr)

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JP2011502467A (ja) 2011-01-27
ES2520025T3 (es) 2014-11-11
CA2666758A1 (fr) 2008-04-24
CA2666758C (fr) 2013-06-18
US20170369856A1 (en) 2017-12-28
HK1133038A1 (en) 2010-03-12
US20090280539A1 (en) 2009-11-12
US9102924B2 (en) 2015-08-11
DK2079834T3 (en) 2014-11-24
US8962293B2 (en) 2015-02-24
CN101528919B (zh) 2014-07-23
US9738876B2 (en) 2017-08-22
CN101528919A (zh) 2009-09-09
US20150218537A1 (en) 2015-08-06
US20090148891A1 (en) 2009-06-11
EP2079834B1 (fr) 2014-08-20
WO2008046612B1 (fr) 2008-06-12
US10035993B2 (en) 2018-07-31
WO2008046612A1 (fr) 2008-04-24
JP5189101B2 (ja) 2013-04-24

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